Abstract
Hemp (Cannabis sativa L.) is commonly grown for the medicinal secondary metabolites produced by pistillate inflorescences. Micropropagation is a valuable method of propagating hemp plants because of the aseptic process and the production of true-to-type propagules. The hemp cultivar TJ’s CBD was used for a series of experiments to compare media inputs and practices for the clonal micropropagation of hemp. For stage I, shoot tips harvested from stock plants that were grown in a growth chamber produced less endogenous contamination in newly established cultures than shoot tips harvested from the greenhouse. In addition, stage I disinfection treatments with 20%, 40%, and 60% bleach (7.5% sodium hypochlorite) for 10 minutes had no differences in surface contamination rates. All concentrations were able to clean explants equally, and no damage to the explants was observed. For stage II, there were no differences in growth and multiplication rate between shoot tip or nodal explants. In addition, no differences were observed between the gelling agent’s agar, agargellan, and gellan gum at standard rates. When basal nutrient formulations were compared at standard rates and with their respective vitamins, Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium. Media pH levels of 4.0, 5.0, 5.8, 6.0, and 7.0 were compared, and no differences were observed in final fresh weights, shoot lengths, or quality ratings. The pH levels of 5.8, 6.0, and 7.0 generated a greater number of lateral nodes. Sucrose levels of 0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) were also compared, with the 1.5% and 3.0% rates showing greater fresh weights, shoot lengths, and quality ratings. Growth room temperatures of 22, 24, 26, and 28 °C were compared, with temperatures of 28 and 26 °C generating greater fresh weights, shoot lengths, numbers of nodes, and quality ratings compared with cooler temperatures. The cytokinins 6-enzylaminopurine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) were compared at 1.0, 5.0, and 10.0 μM concentrations. The 5.0-μM TDZ treatment generated greater fresh weights and numbers of lateral nodes; however, it also produced the shortest shoot lengths and lowest quality ratings. The 2iP treatments at 1.0 and 5.0 μM, and the BA treatment at 1.0 μM produced the greatest quality ratings. The 5.0-μM 2iP level was considered the best treatment for stage II multiplication based on high ratings, in addition to the greater final fresh weights, shoot lengths, and numbers of nodes that were produced. For stage III experiments, the auxins indole 3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) were compared at concentrations of 0.25, 0.5, and 2.5 μM. Auxin treatments of 0.25 μM NAA, 0.5 μM NAA, and 2.5 μM IBA generated the greatest final shoot fresh weights, root fresh weights, and numbers of nodes. However, the 2.5-μM IBA treatment resulted in a higher overall rating. For stage IV, ex vitro rooting and acclimation trials compared a dome and an intermittent mist system, as well as treated the unrooted cuttings with an externally applied auxin. Acclimating with a dome produced greater shoot heights, fresh shoot weights, and overall ratings compared with the mist system. The auxin treatment mildly increased fresh root weight, but was not as important to acclimation success as the domed environment. It has been concluded that a micropropagation system that uses lower rates of sucrose, higher growing temperatures, and lower rates of the cytokinins BA and 2iP are optimal for the micropropagation of hemp. In addition, when acclimating hemp plants from tissue culture, an in vitro stage III can be bypassed and plants can be rooted ex vitro during stage IV acclimation with a dome with or without additional auxin treatments.
Hemp (Cannabis sativa L.) is a herbaceous annual plant that is commonly cultivated for its desirable secondary metabolites, such as cannabinoids and terpenes. Hemp has been farmed by civilizations around the world for thousands of years (Clarke and Merlin 2013; McPartland et al. 2019). In the United States, hemp is defined under the 2018 Farm Bill as any Cannabis plant, or derivative thereof, that contains not more than 0.3% delta-9 tetrahydrocannabinol on a dry-weight basis (US House of Representatives 2018). This plant is used for several purposes, including food, fiber, and, most notably, its medicinal properties produced by the trichomes found in greatest abundance on pistillate inflorescences (Bernstein et al. 2019; Small and Marcus 2002). Hemp is predominantly dioecious—meaning, staminate and pistillate inflorescences develop on separate plants (Punja et al. 2017). This outcrossing reproductive habit results in a high level of genetic recombination in the progeny. Thus, it is common practice to use asexual propagation to produce and maintain entirely female clones of Cannabis (Clarke 1993). Growing this plant directly from seed poses a potential risk for growers because the segregation of traits often causes inconsistent vegetative growth and flowering in the field or greenhouse. Feminization of seeds can be induced by hormone manipulation (Mohan Ram and Sett 1982); however, there is still the possibility of growing a staminate plant inadvertently. As an anemophilous species, staminate flowers have substantial pollen production that may fertilize pistillate inflorescences and can result in significantly reduced secondary metabolite production. In addition, asexual propagation helps maintain disease-free plants. There are many pathogens that attack hemp, such as hops latent viroid, Fusarium oxysporum, and powdery mildew (Golovinomyces cichoracearum); if infected, these pathogens can reduce plant quality and yield significantly (Punja et al. 2017; Warren et al. 2019).
Plant tissue culture is the growth of plants on a sterile, nutrient medium in vitro under controlled environmental conditions (Kyte et al. 2013). Micropropagation is one of the valuable tissue culture techniques used to propagate plant material asexually. This in vitro process of cloning has been used by the horticulture industry for several decades. Micropropagation is preferable over traditional vegetative cuttings for the propagation of high-value crops as a result of its aseptic nature, efficient use of space, and vigorous propagation rate (Kyte et al. 2013). Micropropagation is often described as consisting of five distinct stages (Debergh and Maene 1981). Stage 0 involves the selection and growth of stock plants for the purpose of harvesting explants before the stage I disinfection and establishment phase (Debergh and Read 1991). Stage I involves the establishment of aseptic cultures often achieved with the use of a disinfecting agent to disinfect and achieve explant surface sterility. Stage II is the multiplication phase during which plantlets are grown and subdivided until the desired number of propagules are attained. Stage III is the period in which root induction occurs, and stage IV is when plants are acclimatized and finally transitioned to the environment in which they will be grown. Different species have varying and often specific requirements for efficient micropropagation. In addition, there are many variables that can be optimized at each stage, including disinfection protocols, media inputs, and environmental conditions such as temperature and light. Currently, well-defined micropropagation protocols that lead consistently to successful micropropagation in hemp are lacking. Therefore, the objective of our research was to evaluate various media inputs and practices, and to highlight considerations that optimize the stages of hemp micropropagation. Trials were conducted to evaluate the factors that affect contamination rate and establishment success during stage I. To optimize stage II multiplication more efficiently, explant growth was compared on several commercial basal nutrient formulations, with different explant types, varying concentrations of sucrose, adjustments to the pH of the media, various growing temperatures, different types of gelling agent, and several cytokinins and concentrations on explant growth and development. For stage III, auxin types and concentrations were evaluated on explant growth and rooting success. Last, for stage IV, factors that affect ex vitro establishment and acclimation were examined. The information presented serves as a foundation for the successful micropropagation of hemp.
Materials and Methods
Plant material
The hemp cultivar TJ’s CBD was used for all experiments. Clonal stock plants of ‘TJ’s CBD’ hemp were maintained at the Kenneth Post Laboratory Greenhouses at Cornell University in Ithaca, NY, USA. Stock plants were maintained in a greenhouse with average 26/20 °C day-/nighttime temperatures with supplemental high-pressure sodium lighting to provide light 18 h⋅d–1 for vegetative growth. Plants were grown in 19-L containers with Lambert® LM-111 all-purpose mix (Lambert, Rivière-Ouelle, CA, USA), and were fertigated daily with 150 mg⋅L–1 N of 15.0–2.2–12.5 (Jack’s Professional® 15–5–15 CA-MG LX). For stage I experiment, stock plants maintained in the growth chamber were propagated from an initial batch of micropropagated shoot tips first disinfected from greenhouse stock material. This was done to ensure the growth chamber stock plants started without any pests, diseases, or endogenous contaminants. The chamber was maintained at an average of 25/21 °C day/night temperatures with supplemental T5 fluorescent lighting to provide light 18 h⋅d–1 for vegetative growth at ∼600 µmol⋅m–2⋅s–1 photosynthetically active radiation (PAR). The container size, soilless mix, and fertigation practices remained the same except with less frequent waterings.
Culture media and growing conditions
Many of the media components used for these experiments were purchased from PhytoTechnology Laboratories® (Shawnee Mission, KS, USA); product numbers are included for reference. Unless otherwise mentioned, the basal medium used for growing stock plants and subsequent experiments consisted of Murashige and Skoog (MS) basal medium with vitamins (product ID M519) (Murashige and Skoog 1962). After the addition of MS and sucrose, the pH of the medium was adjusted to 5.8 with either 0.1 N potassium hydroxide or 0.1 N hydrochloric acid, and the medium was solidified with the addition of 0.7% (wt/vol) agar (product ID A296). Culture vessels were autoclaved at 121 °C and 100 kPa for 20 min. No plant growth regulators were used for stock plants or in any experiment unless otherwise stated. No antibiotics or microbial suppressants were used in any media. For the stage I trial, disposable 16 × 150-mm culture tubes (Fisherbrand® catalog no. 14-961-31; Fisher Scientific, Hampton, NH, USA) were used, each containing 5 mL of media. For stage II and III trials, explants were cultured in Magenta™ GA-7 vessels (Magenta LLC, Lockport, IL, USA) containing 60 mL of media and capped with nonventilated lids. For trials that used Magenta GA-7 vessels, each vessel contained three 1-cm single-node explants of uniform size unless otherwise stated. Cultures were maintained in a growth room at ∼25 °C under a 16/8-h light photoperiod at 100 μmol⋅m–2⋅s–1 PAR provided by T5 fluorescent 4100k white lights (Sylvania® FP28/26W/841/SS/ECO).
Statistical analysis
Data for all experiments were analyzed using R statistical software (R Foundation for Statistical Computing, Vienna, Austria). A linear mixed-model analysis was used for all trials with the lme4 package (Bates et al. 2015). Data for all experiments were collected on a per-explant basis, with explant being the unit of replication. For trials that were carried out using Magenta GA-7 vessels, three uniform nodal explants were cultured in each vessel. For these trials, treatments were considered a fixed effect, whereas the effect of culture vessels was considered a random effect. For experiments that featured trial repetitions over time, trial was also included in the models as a random effect. In all cases, the effect of trial was negligible, and the simpler mixed model was used to maintain uniformity among trials. The interaction terms were evaluated for the stage I (stock environment × bleach concentration) and stage IV (propagation environment × auxin) factorial designs, but neither was significant, and model simplification was used to present the results more effectively. Post hoc analysis was carried out by using the emmeans package (R Foundation for Statistical Computing, Vienna, Austria) to compare estimated marginal mean using Tukey’s honestly significant difference (HSD) at P < 0.05 for mean separation. For the stage I trial, data were collected as a binary response where 1 = contaminated and 0 = not contaminated. Data for this trial were analyzed with a generalized linear model with a binomial distribution. Environment and bleach concentration were analyzed as fixed factors, with main effects compared using Tukey’s HSD at P < 0.05 for mean separation.
Stage I: Initiation
Two experimental factors, donor plant environment and bleach concentration, were tested for their effect on disinfection and establishment success. Explants were either harvested as traditional cuttings from greenhouse-maintained stock plants or from stock plants grown from sterile cultures and established in an isolated growth chamber. Three disinfection treatments were tested by soaking cuttings in Clorox® [7.5% sodium hypochlorite (NaOCl)] bleach solutions diluted to either 20%, 40%, or 60% of the initial concentration. All disinfection solutions contained 0.1% Tween-20 as a surfactant. On the day that cultures were initiated, fresh shoot tips several centimeters in length were harvested from the stock plants, leaves were removed, and explants were placed immediately into 946-mL wide-mouth Mason jars with tap water to prevent dehydration. There were 20 shoot tips harvested per treatment at each initiation date from both the greenhouse and growth chamber. Cuttings were then taken to the laboratory, where the jars were covered with cheesecloth and secured by the jar ring. The jars were then placed under lukewarm, running water for 10 min as a pretreatment to remove surface debris. Immediately after the pretreatment, all 20 cuttings were transferred to an autoclaved 500-mL glass bottle, where they were disinfected with 300 mL of a respective disinfection solution. Cuttings were disinfected for 10 min while on an orbital shaker at 200 rpm. After the 10 min, explants were triple-rinsed with sterile water for 1 min each rinse to remove residual bleach. To avoid dehydration, explants remained in the third rinse of sterile water until they were cultured. When explants were removed from their holding water, they were given a fresh cut with a sterile scalpel to 1 cm in length. Explants were then placed immediately in individual 16 × 150-mm culture tubes containing 5 mL of half-strength MS medium including vitamins, 1.5% sucrose (wt/vol), and 0.7% agar (wt/vol) (PhytoTechnology Laboratories, A296) at pH of 5.8. Shoot tips were assessed after 30 d for mortality and contamination.
Stage II: Multiplication
Numerous trials were conducted looking at the effect of different media inputs on explant growth and productivity. Unless otherwise stated, each trial was replicated three times with five vessels per treatment (n = 15 per trial, N = 45 total). Each vessel contained three 1-cm single-node explants of uniform size. After 30 d in culture, fresh weights were taken and explants were placed into fresh media without subdivision. After 60 d in culture, data collected included explant fresh weight, length of the largest shoot, number of lateral nodes, and a subjective quality rating of 1 to 5 (Fig. 1). Quality ratings were defined with a rating of 5 being the highest and representing plants that appeared green, healthy, and exhibited greater vigor, height, and rooting, and the ability to be multiplied into several explants. A rating of 1 was the lowest; these plants were defined as having an unhealthy appearance with abnormal or stunted growth; the presence of chlorosis, necrosis, or hyperhydricity; and exhibited lesser vigor, height, and rooting, with no ability to be multiplied (Fig. 1).
Quality rating system used for in vitro trials on Cannabis sativa L. Quality ratings were defined such that a rating of 5 was the highest and represent plants that appear green and healthy; exhibit greater vigor, height, and rooting; and have the ability to be multiplied into several explants. A rating of 1 was the lowest; these plants were defined as having an unhealthy appearance with abnormal or stunted growth; the presence of chlorosis, necrosis, or hyperhydricity; exhibited lesser vigor, height, rooting; and had no ability to be multiplied.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Sucrose.
To determine the optimal sucrose concentration for the medium, rates of 0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) were compared. The trial was repeated two times (n = 15 per trial, N = 30 total), and data collected included all discussed previously, except rooting and number of nodes. This trial began with 1-cm shoot tips.
Basal media.
Several commercial basal nutrient formulations were tested for their effect of in vitro hemp explant growth. These included MS medium (Murashige and Skoog 1962) (PhytoTech, product ID M519), Linsmaier & Skoog (LS) medium (Linsmaier and Skoog 1965) (PhytoTech, product ID L689), Driver & Kuniyuki Walnut (DKW) medium (Driver and Kuniyuki 1984; McGranahan et al. 1987) (PhytoTech, product ID D2470), and Lloyd & McCown Woody Plant Medium (WPM) (Lloyd and McCown 1980) (PhytoTech, product ID L449), all with their respective vitamin compositions. Each medium was evaluated at their recommended rates of 4.43, 4.43, 5.32, and 2.41 g⋅L–1, respectively. The trial was repeated three times (n = 15 per trial, N = 45 total) using 1-cm shoot tips, and data collected included all discussed previously, along with a count of the number of shoots produced by each explant.
Gelling agent.
Several types of gelling agents were evaluated for their effect on explant growth, including agar (PhytoTech, product ID A296), agargellan (PhytoTech, product ID A133), and gellan gum (PhytoTech, product ID G434) at their respective middle recommended rates of 0.7%, 0.4%, and 0.2% (wt/vol), respectively. The trial was repeated three times (n = 15 per trial, N = 45 total), and data collected included all discussed previously.
Media pH.
Media pH were also evaluated for their effect on explant growth. Before autoclaving, the media were adjusted to a pH of 4.0, 5.0, 5.8, 6.0, or 7.0. The trial was repeated once, with 10 replicate vessels per treatment and three explants per vessel (n = 30). Data collected included all discussed previously.
Explant type.
Shoot tips and single nodes were compared as explant types for their effect on in vitro propagation. The trial was repeated once, with 15 replicate vessels per treatment and three explants per vessel (n = 45). Data collected included all discussed previously, but were only collected at the 30-d mark.
Temperature.
The temperature of the growing environment for stage II propagules of hemp was evaluated at 22, 24, 26, and 28 °C. The trial was repeated three times (n = 15 per trial, N = 45 total), and data collected included all discussed previously.
Cytokinin.
The effect of various cytokinins at different concentrations were evaluated for their effect on explant growth. The cytokinins evaluated included 6-benzyladenine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ). Each cytokinin type was evaluated at 1.0-, 5.0-, and 10.0-μM concentrations. An additional control with no growth regulator was also included in the trial. The trial was repeated three times (n = 15 per trial, N = 45 total), and data collected included all discussed previously, along with a count of the number of shoots generated by each explant.
Stage III: In vitro rooting
The auxins indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) were tested at rates of 0.25, 0.5, and 2.5 μM. In addition, a treatment with no growth regulator was included in the trial as a negative control. The trial was repeated three times (n = 15 per trial, N = 45 total), and data collected included all discussed previously. Root fresh weight for each explant was recorded by removing roots carefully with a scalpel, submerging in warm water to eliminate excess agar, and dabbing on a dry paper towel three times before weighing.
Stage IV: Ex vitro rooting and acclimation
An acclimation study was conducted by harvesting 2.5-cm-long shoot tips from culture and rooting ex vitro in a greenhouse. Explants were acclimated either by establishing in a polyethylene humidity dome or with an intermittent mist bench. In addition, harvested explants were either transplanted with no rooting stimulant or were treated with a 0.1% IBA rooting powder (Hormex No.1; Hormex, Westlake Village, CA, USA). The trial was designed and analyzed as a factorial design (environment × auxin) with each treatment combination involving 30 shoot tips. For each treatment, shoot tips were removed aseptically and placed into jars containing tap water to prevent desiccation. Shoot tips were transplanted into two 72-count trays containing 50% Lambert LM-111 all-purpose mix and 50% course perlite. Half the cuttings in each tray were planted untreated, whereas the second half were treated with the rooting powder. When ready, one tray was covered with a humidity dome, whereas the other was exposed to intermittent mist. Both trays were placed under intermittent mist to standardize the environment across treatments. The intermittent mist bench was shaded with a 30% shadecloth applied above the misters. Trays were also placed on a heat mat to maintain a rhizosphere temperature of 28 °C. The intermittent mist system was set with frequency of 20 min, with a duration of 4 s. On average, air temperature was maintained at 25 °C, and plants received 300 to 500 μmol⋅m–2⋅s–1 light. Data were collected after 14 d, at which point individual plants were removed from the trays and their roots washed carefully. Before weighing, washed roots were dabbed on a dry paper towel three times. Data collected included fresh shoot and root weight, plant height, and a quality rating of 1 to 5. A similar rating scale was adopted as the one described for the in vitro trials, where a rating of 5 was the highest and represented plants that appeared green, healthy, and exhibited greater vigor, height, and rooting (Fig. 1). A rating of 1 was the lowest; these plants were defined as having an unhealthy appearance with abnormal or stunted growth, the presence of chlorosis or necrosis, and plants that exhibited lesser vigor and height, and no rooting (Fig. 1).
Results
Stage I: Initiation
Donor plant environment and bleach concentration were evaluated for their effects on disinfection and establishment success. Results showed significant differences in the rate of contamination between explants harvested from stock plants maintained in a growth chamber vs. the greenhouse (P < 0.001) (Table 1). Explants harvested from the growth chamber resulted in less contamination (1.7%) when compared with explants harvested from plants maintained in a greenhouse (88.3%) (Table 1). No significant differences were observed between bleach concentration treatments (Table 1). Disinfection with 20%, 40%, and 60% bleach (7.5% NaOCl) for 10 min resulted in 45.0%, 47.5%, and 42.5% contaminated cultures, respectively (Table 1).
Stage I results comparing stock plant grow environment (greenhouse or growth chamber) and disinfection with 20%, 40%, or 60% bleach (7.5% NaOCl) on Cannabis sativa L. ‘TJ’s CBD’ explant contamination and mortality.
Stage II: Multiplication
Sucrose.
Sucrose concentrations of 0.0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) were evaluated for their effect on explant growth. Significant differences were found between sucrose concentrations on 60-d fresh weights (P < 0.001), shoot lengths (P < 0.001), and quality ratings (P < 0.001) (Table 2). Sucrose treatments of 1.5% and 3.0% resulted in greater 60-d fresh weights, shoot lengths, and quality ratings when compared with the other concentrations (Table 2). The 0%, 4.5%, and 6.0% sucrose treatments expressed no difference in 60-d fresh weight and length; however, 6.0% had the lowest explant quality of all treatments (Table 2).
Stage II results comparing the effects of sucrose concentration added to the tissue culture media on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Basal media.
Several commercial basal nutrient formulations, and their respective vitamin compositions, were evaluated for their effect of explant growth. Significant differences were observed between media formulations on 60-d fresh weights (P < 0.001), shoot lengths (P = 0.017), numbers of shoots (P < 0.001), and quality ratings (P = 0.002) (Table 3). Explants that were cultured in MS and LS media had the greatest 60-d fresh weights and numbers of shoots (Table 3). Explants cultured in MS, LS, and DKW media showed comparable shoot lengths and ratings; however, the DKW treatment also showed similar shoot lengths and ratings to WPM (Table 3). Overall, explants cultured in DKW and WPM had the lowest 60-d fresh weights and numbers of shoots, and the lowest quality ratings (Table 3).
Stage II results comparing Murashige and Skoog (MS), Linsmaier & Skoog (LS), Driver & Kuniyuki Walnut Media (DKW), and Lloyd & McCown Woody Plant Medium (WPM) at standard rates on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Gelling agent.
Several types of gelling agents, at their intermediate recommended rates, were evaluated for their effect on explant growth. There were no significant differences between agar, agargellan, and gellan gum on explant 60-d fresh weights (P = 0.065), shoot lengths (P = 0.798), numbers of nodes (P = 0.768), and explant quality ratings (P = 0.804) (data not shown). The incidence of rooting was also recorded, but no differences were observed, with 91.0%, 96.0%, and 93.0% of explants producing roots in the agar, agargellan, and gellan gum treatments, respectively.
Media pH.
Media pH was also evaluated for its effect on explant growth during stage II. Significant differences between pH treatments were observed for the number of lateral nodes (P < 0.001) and quality ratings (P = 0.015), but no differences were observed for 60-d fresh weights (P = 0.098) and shoot lengths (P = 0.483) (Table 4). None of the pH treatments expressed differences in 60-d fresh weights or shoot lengths; however, pH 5.8, 6.0, and 7.0 produced the greatest numbers of nodes among treatment groups (Table 4). The quality ratings of explants in the 5.8 pH treatment was found to be greater than 4.0 pH. Quality ratings for the 5.0, 6.0, and 7.0 pH treatments were intermediate and did not differ from either the 5.8 or 4.0 pH treatments (Table 4). The incidence of rooting was also recorded, but no differences were observed with 86.7%, 83.3%, 88.0%, 82.8%, and 79.3% of explants producing roots in the 4.0, 5.0, 5.8, 6.0, and 7.0 pH treatments, respectively.
Stage II results comparing the effects of medium pH on the growth of Cannabis sativa L.‘TJ’s CBD’ explants after 60 d in culture.
Explant type.
Shoot tips and single-node stem segments were compared as explant propagules for their effect on propagation productivity. After 30 d in culture, shoot tips and nodal explants did not differ in either fresh weight (P = 0.740), shoot length (P = 0.501), number of nodes (P = 0.302), or quality rating (P = 0.523) (data not shown). No differences were observed for the number of explants that produced roots, with 60.0% of shoot tips and 57.8% of nodal explants producing roots.
Temperature.
The temperature of the growing environment was evaluated for its effect on explant growth. Significant differences were found between temperature treatment on 60-d fresh weights (P < 0.001), shoot lengths (P < 0.001), numbers of lateral nodes (P < 0.001), numbers of roots (P < 0.001), and quality ratings (P < 0.001) (Table 5). Grow-room temperatures of 28 °C and 26 °C resulted in the greatest 60-d fresh weights, numbers of nodes, and quality ratings compared with cooler temperatures of 24 and 22 °C (Table 5). The 26 °C treatment was intermediate in shoot length and did not differ from either the 28 or 24 °C treatments. The 24 and 22 °C treatments produced similar fresh weights; however, the 22 °C treatment showed the smallest shoot lengths, least numbers of nodes, and lowest explant quality of all temperature treatments (Table 5).
Stage II results comparing temperatures 22, 24, 26, and 28 °C on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Cytokinin.
Three cytokinins—BA, 2iP, and TDZ—at concentrations of 1.0, 5.0, and 10.0 μM, were evaluated for their effect on explant growth and productivity. Significant differences were found between cytokinin treatments on 60-d fresh weights (P < 0.001), shoot lengths (P < 0.001), numbers of shoots (P < 0.001), numbers of lateral nodes (P < 0.001), and quality ratings (P < 0.001) (Fig. 2). Explants that were cultured in media containing 5.0 and 10 μM TDZ produced the greatest 60-d fresh weights, numbers of shoots, and numbers of nodes, with the 5.0-μM TDZ treatment being significantly higher than the 10-μM TDZ treatment (Fig. 2A, C, and D). However, it was also observed that the 5.0- and 10-μM TDZ treatments had significantly smaller shoot lengths and the lowest explant ratings (Fig. 2B and E). The greatest shoot lengths and quality ratings were observed among the BA and 2iP treatments, followed closely by the control (Fig. 2B and E). Rooting was also monitored between cytokinin treatments, with all cytokinin treatments showing significantly less explants with roots than the control (Fig. 2F). However, explants cultured in media containing BA or TDZ had significantly less explants with roots than those cultured in 2iP (Fig. 2F).
Stage II results comparing the cytokinins 6-benzyladenine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) at concentrations of 1.0, 5.0, and 10.0 µM on the micropropagation of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture. Mean separation within columns is indicated by letters using Tukey’s honestly significant difference at P < 0.05.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Stage III: In vitro rooting
The auxins IBA and NAA were evaluated at rates of 0.25, 0.5, and 2.5 μM for their effect on explant rooting. Significant differences were found between auxin treatments on 60-d fresh weights (P < 0.001), root fresh weights (P < 0.001), shoot lengths (P < 0.001), numbers of lateral nodes (P < 0.001), and quality ratings (P < 0.001) (Fig. 3). The 0.25- and 0.5-μM NAA treatments produced the greatest root fresh weights (Fig. 3B), whereas the 0.5-μM NAA treatment also produced the greatest 60-d fresh weights (Fig. 3A). The 0.25- and 0.5-μM NAA treatments, along with the 2.5-μM IBA treatments also had the greatest shoot lengths and produced the greatest numbers of lateral nodes (Fig. 3C and D). However, it was the control and 0.25-μM IBA treatment that showed the highest quality ratings, with the 0.5-μM NAA, 2.5-μM IBA, 0.5-μM NAA, and 0.5-μM NAA treatments showing intermediate quality ratings (Fig. 3E).
Stage III trial results comparing the auxins indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) at concentrations of 0.25, 0.5, and 2.5 µM on the rooting of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture. Mean separation within columns indicated by letters using Tukey’s honestly significant difference at P < 0.05.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Stage IV: Ex vitro rooting and acclimation
An acclimation study was conducted to compare propagation environment (intermittent mist system vs. humidity dome) and auxin treatment on the rooting and acclimatization of microshoots harvested from in vitro stock plants. Significant differences were observed between acclimation environments for shoot weights (P < 0.001), shoot heights (P < 0.001), and quality ratings (P < 0.001), but not between auxin treatment (P = 0.132, P = 0.825, P = 0.538, respectively), or the interaction between environment and auxin treatment (P = 0.474, P = 0.229, P = 0.798, respectively). Significant differences between acclimation environments (P = 0.0203) and auxin treatments (P < 0.001) were observed for root weights, but the interaction was still not significant (P = 0.377). Acclimating shoots with a humidity dome produced hemp plants with greater shoot heights, fresh shoot and root weights, and quality ratings compared with those acclimated through an intermittent mist system (Table 6). No significant differences in shoot weights, shoot heights, and quality ratings were observed between explants transplanted with no rooting stimulant or those treated with a 0.1% IBA rooting powder (Table 6). A difference was observed in root weights, with explants treated with 0.1% IBA rooting powder having greater root weights than those not treated (Table 6).
Stage IV results comparing propagation environment (intermittent mist system vs. humidity dome) and auxin treatment on the acclimatization of 2.5-cm Cannabis sativa L. ‘TJ’s CBD’ shoots.
Discussion
Stage I: Initiation
Stage I disinfection and establishment is one of the most challenging aspects of micropropagation. A stage I protocol that is both simple and effective can reduce the time it takes to introduce new and desired genetics in vitro. Donor plant environment and bleach concentration were evaluated because they are critical to disinfection and establishment success (Debergh and Read 1991). When stock plants were grown in a greenhouse vs. a growth chamber, there was a lower stage I success rate (Table 1). In these experiments, almost every contaminated culture was caused by endophytic microbes, not surface contamination (data not shown). In most of these cases, the unidentified contaminants were white and exuded from the vascular tissue at the proximal end of the explant several days after initiation. Explants harvested from stock plants grown in the growth chamber expressed virtually no contamination (Table 1). This stock material was isolated from the pathogens and pests that were present in the greenhouse, and consequently was cleaner at the beginning of stage I. These results illustrate the importance of what is referred to as stage 0, or starting with clean stock material, and maintaining plants in a clean, controlled environment (Debergh and Read 1991). Results suggest that bleach concentration had less impact on contamination rate than the growing environment (Table 1). However, little to no surface contaminants (either bacterial or fungal) were observed, which suggests that without the presence of the endophytes, the bleach treatments would have been significantly more effective. Last, even the highest rate of bleach (60% of a 7.5% NaOCl solution for 10 min) did not affect significantly explant survival negatively, which suggests that higher concentrations may be possible and may be more effective without excessive damage to explants.
Stage II: Multiplication
Sucrose.
Sucrose concentration was one of the factors that affected hemp explant growth in vitro most significantly. It was observed that sucrose treatments of 1.5% and 3.0% resulted in greater 60-d fresh weight, shoot length, and quality rating when compared with the other sucrose concentrations (Table 2, Fig. 4). The treatment with no sucrose consistently had one explant in each vessel (33%) that performed adequately, whereas the other two explants in the vessel remained stunted. This suggests competition for nutrients in vitro may play a role in performance. The results indicate that 1.5% sucrose is the optimal treatment for the micropropagation of hemp ‘TJ’s CBD’. Although there was no statistical difference between the 1.5% and 3.0% treatments, reducing sugar consumption by about half will reduce media input costs. In addition, there was a noticeable reduction of hyperhydricity in plantlets exposed to lower concentrations of sucrose (no data shown). Sucrose concentrations of 4.5% and 6.0% reduced explant quality and growth consistently and considerably (Fig. 4). Sucrose has osmoregulatory properties that may lead to the inability of explants to access water and nutrients at high concentrations. It should be noted that the impact of sucrose concentration on explant growth is dependent on the quality and intensity of light provided during the culture cycle (Eckstein et al. 2012). Commercial laboratories should take care to evaluate a range of 1.5% to 3.0% sucrose to optimize most effectively against their specific growing environments.
Comparison of 0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) sucrose concentrations on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Basal media.
From the commercial media formulations evaluated in this trial, MS and LS showed no significant difference; however, LS had slightly higher average shoot weight, shoot length, and number of shoots (Table 3, Fig. 5). A large difference between MS and LS is the vitamin composition of each medium, which suggests that further optimizations can be made by changing the vitamin composition. Explants grown on DKW medium were adequate, but more brittle, and more likely to express interveinal chlorosis compared with the MS and LS treatments (Fig. 5). This differs from the findings by Page et al. (2021), who found that DKW medium is better than MS for in vitro growth and multiplication of hemp. The contrast in results may be a result of differences in the genetics or environmental conditions used in the study. Shoots grown on WPM were noticeably stunted, which is likely a result of the reduced amount of nutrients available or the nitrogen sources used in this medium (Fig. 5). In addition, explants in WPM also started developing pistils by the end of the trial, which is likely a stress-induced flowering response. These results suggest that hemp favors high-nutrient media such as MS, LS, and DKW. Although these results show that LS and MS media may be interchangeable, the use of MS medium may be preferred because it is widely used and readily available.
Comparison of Driver & Kuniyuki Walnut medium (DKW), Murashige and Skoog (MS), Linsmaier & Skoog (LS), and Lloyd & McCown Woody Plant Medium (WPM) at standard rates and with respective vitamins on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Gelling agent.
Evaluation of hemp growth in agar, agargellan, and gellan gum at standard rates revealed no statistical differences between treatments (data not shown). For gelling agents, the lack of difference between treatments indicates that gelling agents themselves may not be as important as the concentration at which they are used. Higher concentrations of gelling agents may reduce hyperhydricity and improve explant quality. Because of its high viscosity, gellan gum is often used at rates of 2 to 4 g⋅L–1 less than the 6 to 10 g⋅L–1 recommended for agar (Hartmann et al. 2018) (PhytoTechnology Laboratories). For plants susceptible to hyperhydricity, gellan gum is often not recommended (Hartmann et al. 2018). Although not observed during our experiment, other stock cultures grown on gellan gum have shown a decline in stock explant quality and hyperhydricity after prolonged exposure (data not shown). The effects of gellan gum and similar products on hemp growth and multiplication needs to be evaluated further.
Media pH.
Evaluation of media pH for its effect on explant growth showed no differences in fresh weights and shoot lengths; however, the 5.8, 6.0, and 7.0 pH treatments generated more nodes and appeared less chlorotic than the 4.0 and 5.0 pH treatments (Table 4). Slight improvements in explant quality, and the ease of pH adjustment, indicate that a standard pH of 5.8 is preferred for micropropagation of hemp.
Explant type.
Comparison of shoot tip and single-node explants revealed no differences in growth and productivity (data not shown). The lack of statistical difference is beneficial because it suggests that both explant types are suitable for stage II multiplication. A noticeable trend throughout all trials was that explants that had developed roots were larger, higher quality, and less chlorotic than those that did not. An observation was made that single nodes that were taken from the proximal base of explants appeared to have a higher rate of stunted growth compared with nodes farther up the stem.
Temperature.
In any plant production system, the temperature of the growing environment is an important factor to consider. The results from this trial showed significant differences in explant growth and productivity with increasing temperature (Table 5, Fig. 6). Overall, explants grown at 28 °C grew considerably faster and taller, produced more nodes, and were of higher quality (Table 5, Fig. 6). In addition, greater root formation was observed with increasing temperatures. These results were expected because it is known that the growth of hemp is enhanced in warmer environments and stunted in colder climates (Chandra et al. 2020; Galic et al. 2022).
Comparison of temperatures 28, 26, 24, and 22 °C on the growth of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Cytokinin.
Stage II multiplication often relies on added growth regulators to promote explant growth and differentiation. For the cytokinin trial, BA, 2iP, and TDZ were compared at 1.0, 5.0, and 10.0 μM concentrations. The 5.0-μM TDZ treatment generated explants with the greatest fresh weights and the greatest numbers of shoots and lateral nodes per explant (Fig. 2A, C, and D). Although data were not collected, it was also observed that TDZ at all levels resulted in greater hyperhydricity compared with BA and 2iP at the same levels. In addition, considerable callus formation was observed on explants when TDZ was in the medium. This has been illustrated in other species at relatively low concentrations (Huetteman and Preece 1993). Callus is not a desirable growth response during stage II of micropropagation because of the increased threat of cell mutations. Other publications have recommended the use of TDZ for hemp micropropagation (Lata et al. 2009; Wang et al. 2009); however, the data from these experiments do not support that conclusion. The number of shoots and nodes was increased by using the two highest concentrations of TDZ, but the shoots were shorter and the quality of the plants was lower (Fig. 2B and E). On the other hand, BA at 1.0 μM resulted in noticeably more consistent explants, with greater height, more shoots, and more nodes than the control (Fig. 2B–D). Explants cultured in 5.0 μM BA produced even more shoots and nodes, but explant growth became inconsistent and chlorotic, and with greater callus formation than the lower BA treatment (Fig. 7). The 10.0-μM BA treatment resulted in a substantial decline in explant quality defined by stunting and significant chlorosis (Fig. 7). The 1.0-μM 2iP treatment performed almost identically to the 1.0-μM BA treatment; however, 2iP at 5.0 μM produced greener, higher quality explants compared with BA at the same rate (Figs. 2 and 7). At 10.0 μM 2iP, explants did not experience the same decline in quality compared with other cytokinins tested at the same concentration (Fig. 7). Explants cultured in 2iP also exhibited the least amount of callus formation across treatments relative to BA and TDZ, and retained noticeable root formation at the low and medium rates. These results support the use of BA or 2iP between concentrations of 1.0 and 5.0 μM for the micropropagation of hemp at stage II. Research by Grulichová et al. (2017) also indicated that low rates of plant growth regulators produce hemp plantlets with greater vigor. The cytokinin meta-topolin (mT) has been recommended as the superior cytokinin for multiplication in other hemp micropropagation publications (Grulichová et al. 2017; Lata et al. 2016; Mestinšek Mubi et al. 2020). There are no known published reports of 2iP being tested for the purpose of hemp micropropagation. Comparison of 2iP and mT is an opportunity for future experimentation.
Comparison of the cytokinins 6-benzyladenine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) at concentrations of 1.0, 5.0, and 10.0 µM on the micropropagation of Cannabis sativa L. ‘TJ’s CBD’ explants after 60 d in culture. PGR = plant growth regulator.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Stage III: In vitro rooting
Rooting in vitro is an important precursor to stage IV acclimation. In our trial, the auxins IBA and NAA were compared at 0.25-, 0.5-, and 2.5-μM concentrations for their effect on explant rooting. Overall, although 0.25- and 0.5-μM NAA treatments produced the greatest root fresh weights, they also generated large amounts of callus and showed diminished explant quality (Fig. 3). The control with no auxin and 0.25-μM IBA treatment showed adequate root formation and exhibited high-quality ratings (Fig. 3). Research by Mestinšek Mubi et al. (2020) also reported that some explants grown on media without plant growth regulators developed roots. NAA across all treatment levels resulted in considerable callus formation that increased as auxin concentration increased (Fig. 3). Roots developed on media supplemented with NAA became thicker, but were suppressed as concentration increased. NAA at 2.5 μM experienced a considerable reduction in root formation. NAA at 0.20 μM (37.24 μg⋅L–1) was used for rooting explants by Smýkalová et al. (2019), but only a 50% success rate was reported. As IBA treatment levels increased, all measurements gradually increased, except for explant quality (Fig. 3). Roots produced on high IBA media were also finer and longer. Overall, a concentration in the realm of 2.5 μM IBA is optimum for in vitro root induction.
Lata et al. (2009) also found IBA at a concentration of 2.5 μM to be the preferred auxin for in vitro rooting of hemp, with 95% of explants showing adequate rooting.
Stage IV: Ex vitro rooting and acclimation
Acclimation of plantlets grown in vitro is the end goal of any micropropagation program. In our trial, two environments (intermittent mist system vs. humidity dome) were compared. In addition, the benefit of applying an auxin treatment to the shoots was examined for acclimation. Overhead mist produced less-consistent propagules that were more chlorotic, likely a result of nutrient leaching and waterlogging. Plants that were rooted and acclimated under the dome produced plants that were visually superior. Auxin did not play as significant a role as maintaining a proper environment, but did improve rooting mildly (Table 6). A successful combination of these stages has also been shown by Lubell-Brand et al. (2021), which further illustrates the feasibility of ex vitro rooting and acclimation, which may improve production efficiency.
Field trial
During Summer 2021, 100 micropropagated hemp plants were removed from in vitro and acclimated. In early June, these hemp plants were planted outside in the research fields at Cornell University’s Long Island Horticultural Research and Extension Center in Riverhead, NY, USA (Fig. 8). They were planted with rows 18 to 21 m on center and 12 m between plants in-row. The plants all grew uniformly and flowered consistently; no data were collected.
Micropropagated Cannabis sativa L. ‘TJ’s CBD’ flowering uniformly in a research field located at the Long Island Horticultural Research and Extension Center, Riverhead, NY, USA, during Summer 2021.
Citation: HortScience 58, 3; 10.21273/HORTSCI16969-22
Conclusion
Cannabis sativa L. is a genetically diverse species; thus, it is important to consider that different varieties may have unique environmental and media input requirements for optimal micropropagation. This study focused on the hemp cultivar ‘TJ’s CBD’ to eliminate genetic variability as a factor. Commercial facilities should consider evaluating their genetics against different media inputs to optimize productivity more effectively for their specific environmental, genetic, and logistic circumstances. After careful cultivar selection, the first consideration for hemp micropropagation is how to introduce explants efficiently to in vitro culture while limiting contamination. The stage 1 trials illustrate the importance of stock plant management before disinfection. Shoot tips harvested from stock plants maintained in a greenhouse under standard horticultural practices exhibit a significant incidence of endophytic microbes. On the other hand, greater establishment success was observed when stock plants were maintained in a clean and pest-free environment such as a growth chamber. Although bleach concentration appeared to have less impact on contamination rate, it was observed that hemp explants can resist relatively high concentrations of bleach during disinfection. In addition, little to no surface contamination was observed with the bleach concentrations tested (20% to 60% bleach), which suggests these rates are effective at surface-disinfecting explants.
The environmental conditions to which cultures are subjected is as important a consideration as the media inputs used. Our study suggests that a grow-room temperature of 28 °C is optimum for hemp micropropagation. During stage II, nodal and shoot tip explants perform similarly as explant sources. The media MS, LS, and possibly DKW with vitamins and at standard rates are all viable options for hemp micropropagation, but media viability may depend on the specific cultivars being cultured. Maintaining sucrose concentrations between 1.5% and 3.0%, and a media pH of 5.8 or slightly greater is optimum for hemp micropropagation. The type of gelling agent used for hemp micropropagation is less important than other inputs. However, future research is needed to examine the concentrations that are used and the effect of gellan gum on explant quality after long-term exposure.
Although some cultures with no growth regulators experience an acceptable multiplication rate, laboratories may be incentivized to accelerate the multiplication rate further with the addition of cytokinins. Cytokinin type and concentration influences the propagation rate of hemp cultures dramatically. The cytokinins 2iP and BA at rates between 1.0 and 5.0 μM are recommended to improve multiplication rate while maintaining acceptable quality. The use of TDZ and cytokinins at higher concentrations was linked with lower quality and greater incidence of callus growth. For stage III, rooting micropropagated hemp plants in vitro is best achieved by using 2.5 μM IBA. However, these experiments demonstrate that harvesting shoot cuttings from cultures and rooting them ex vitro is largely effective, suggesting that a separate stage III may not be required. It is optimum for commercial laboratories to combine stages III and IV to improve production efficiency and reduce expenses. Rooting hemp shoot tips ex vitro under humidity domes with auxin is preferred over an intermittent mist system. The value of applying auxin at the time of stage III and IV was not significant in our results, but may be beneficial with difficult-to-root varieties.
These experiments examined in detail all five stages of micropropagation for Cannabis sativa L. ‘TJ’s CBD’. This work is the first to investigate comprehensively the complete micropropagation process for hemp. It is important to remember, however, that different species and cultivars have varying requirements during micropropagation to optimize their success. These results form a foundation that can be used to micropropagate hemp successfully.
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